Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23. The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tü494. Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment. This fragment was shown to include the Ptt-resistance promoter. Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations. Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase.